Neuronal induction and migration are fundamental processes in nervous system development, and many human neurological disorders are caused by severe deficiencies in these processes. Our focus is the cranial motor neurons (CMNs), which control vital functions such as chewing, swallowing, and speech in humans. Our long-term goal is to elucidate the cellular and molecular mechanisms underlying the induction and migration of CMNs in the powerful model vertebrate, the zebrafish embryo. Secreted proteins of the sonic hedgehog (Shh) family are essential for vertebrate motor neuron induction. However, little is known about the mechanisms by which Shh signaling pathway components, including the Gli1 and Gli2 transcription factors, specify different types of motor neurons (CMNs and spinal motor neurons (SMNs)) along the anterior-posterior axis of the neural tube. The cellular mechanisms underlying tangential migration of CMNs following induction are equally obscure. Our preliminary studies demonstrate that the zebrafish detour (dtr/gli 1) and you-too (yot/gli2) genes have specific functions in CMN and SMN induction, and that mutations in the trilobite (tri) and valentino (val) genes disrupt particular events during tangential CMN migration. The studies we propose here will define the mechanisms of CMN specification and tangential migration. (1) We will determine whether Gli1 and Gli2 are necessary and sufficient for CMN and SMN induction (a) by characterizing motor neuron development in yot mutants, (b) by determining whether gli1 and gli2 are co-expressed in motor neurons, and (c) by analyzing CMN and SMN induction in dtr; yot double mutants, and following gli1 and gli2 overexpression in wildtype and dtr mutant embryos. (2) We will evaluate the function of Shh pathway components encoded by the chameleon (con) and iguana (igu) genes in CMN and SMN induction. (3) We will determine whether the CMN migration defects of tri and val mutants are caused by specific defects in dynamic cellular behaviors (a) by genetic mosaic analysis of tri mutants, and (b) by analyzing dynamic cellular behaviors of migrating CMNs in wildtype, and tri and val mutant embryos by time-lapse microscopy. The availability of key zebrafish mutants, the ability to employ gain- and loss-of-function approaches, the optical clarity of the zebrafish embryo, and the ability to perform genetic mosaic analysis and time-lapse microscopy represent a powerful combination of tools that will enable us to define the functions of particular genes in CMN development at the cellular and molecular levels. Our studies on motor neuron induction and migration will provide fundamental insights into the mechanisms underlying these processes essential for normal brain development and function.